论文部分内容阅读
目的 利用定量蛋白组学技术探索高糖状态下人视网膜微血管周细胞(HRMPCs)的蛋白表达变化,为糖尿病视网膜病变(DR)早期周细胞缺失的机制研究提供新的蛋白靶点.方法 将HRMPCs分为对照组和高糖组,分别用葡萄糖浓度为25 mmol/L和35 mmol/L的DMEM培养基培养48 h,细胞计数试剂盒-8(CCK-8)法测定各组HRMPCs活性.提取各组细胞的蛋白并进行酶切,得到肽段,取2μg肽段进行飞行时间质谱(TOF-MS)检测,质谱的采集模式为数据依赖性(DDA),对得到的蛋白表达信息进行生物信息学分析.结果 高糖组的HRMPCs数量较对照组明显减少,且高糖组的部分细胞出现体积变小、变形等改变.CCK-8实验结果显示,高糖组HRMPCs吸光度(A450)值为0.75±0.04,明显低于对照组的0.91±0.05,差异有统计学意义(t=5.784,P=0.0002).本研究共鉴定到1972个可定量蛋白,其中差异蛋白有54个(差异倍数>1.5).相比于对照组,高糖组细胞中上调的差异蛋白有13个,包括CTNNB1、CTBP2等,下调的差异蛋白有41个,包括SQSTM1、HMGCS1等.这些差异蛋白主要参与了三羧酸循环、有氧呼吸等生物学过程.结论 高糖刺激HRMPCs会改变多种蛋白的表达,进而影响细胞的呼吸作用和ATP的生成,最终导致周细胞的缺失.“,”Objective To determine the changes of protein expressions in human retinal microvascular pericytes ( HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy ( DR) . Methods HRMPCs were divided into two groups. The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose,while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose. The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin. Peptides of 2μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA. The results were further analyzed using bioinformatics software. Results CCK-8 results showed that the absorbance ( A450 ) of HRMPCs in high glucose group was 0. 75±0. 04,which was significantly lower than 0. 91±0. 05 in control group (t=5. 784,P=0. 0002). In total,1972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1. 5). Among them,13 proteins were up-regulated,including CTNNB1 and CTBP2;while 41 proteins were down-regulated,including SQSTM1 and HMGCS1. The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose,which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.